New targets have been identified by comparative and statistical analysis of healthy and diseased patients, in particular by analyzing tissues and/or blood derived plasma from said patients. Usually, the comparative analysis can be done on different levels, such as on DNA-, RNA-, protein- and post translational levels. One commonly used technique is based on differential gene expression analysis. Briefly, mRNA derived from both, diseased and healthy cells is labeled and subsequently hybridised to a gene chip and quantified. Up- or downregulation of different mRNAs, as derived from the quantification signals, reveals potential new targets. Another approach well known in the prior art is based on the identification and comparison of DNA methylation patterns of DNA molecules derived from healthy and diseased patients.
It is to be noted in the above context, however, that neither the DNA modification (i.e. the DNA methylation pattern) nor the differential gene expression analysis (i.e. the level of mRNA expressed in a cell) necessarily reflects whether a specific protein encoded by the corresponding DNA or the corresponding mRNA is indeed expressed. Therefore, identification of differential expression levels, i.e. quantitative and also qualitative analysis of protein expression patterns of healthy as compared to diseased cells, remains challenging.
A method for identification of differential protein expression levels is based on differential two-dimensional gel analysis of said proteins with subsequent analysis via mass spectrometry, a technique well known to the person skilled in the art. Additionally, methods based on protein fractionation, such as, to mention but a few, techniques based on the use of protein chips, HPLC- and FPLC related techniques which are all known to a person skilled in the art.
Techniques of phage display offer, for example, the possibility to deplete a large library, e.g., an expression library of binding members, on samples, such as tissues or cells that are, for example, derived from a healthy donor, and use the residual population of the library on samples, such as tissues or cells, that are derived, for example, from a diseased donor. Binding members which have been traced by depletion analysis, i.e. which bind to (poly)peptide targets or counterparts of diseased tissues/cells but not to healthy tissues/cells are usually considered to bind to a target which is uniquely (or at least much higher) expressed on the target cells (e.g. the diseased cell). Subsequently, binding member/(poly)peptide target complexes can be identified by, e.g., mass spectrometry or methods for protein analysis well known to the person skilled in the art.
Of particular interest are binding members that internalize upon binding of their target. A person skilled in the art is aware that said binding members can, e.g., then be fused to any substance or any small molecule that might be toxic for the cell thus triggering the killing of the, preferably diseased, cell expressing said target(s), which cell preferably is diseased. As also known in the art, once a target of interest that internalizes has been identified as, e.g., a diseased or cancerous cell, it is then possible to determine further binding members with, e.g., higher affinity to the target and/or higher potential for triggering internalization of said target. Said improved binding members can then be considered, for example, as drugs for treating, e.g., diseased cells expressing said target(s).
To efficiently determine potential targets that have internalized into the cell upon binding of their respective binding member, it would be desirable to separate internalized complexes from non internalized complexes. However, in the prior art, said separation has not been achieved in a qualitative and quantitative satisfying manner thus confronting the skilled artisan with time consuming and complex techniques for determining binding members and targets that internalize.
There is therefore a continuous need to further develop and also ameliorate methods and processes that allow for efficient separation between internalized and non-internalized complexes.